U.S. patent number 10,098,904 [Application Number 15/111,275] was granted by the patent office on 2018-10-16 for zidovudine combination therapies for treating microbial infections.
This patent grant is currently assigned to HELPERBY THERAPEUTICS LIMITED. The grantee listed for this patent is HELPERBY THERAPEUTICS LIMITED. Invention is credited to Anthony Coates, Yanmin Hu.
United States Patent |
10,098,904 |
Coates , et al. |
October 16, 2018 |
Zidovudine combination therapies for treating microbial
infections
Abstract
The present invention relates to the use of a combination of an
anti-retroviral agent such as zidovudine and anti-microbial agents
for killing clinically latent microorganisms associated with
microbial infections and to novel combinations comprising an
anti-retroviral agent such as zidovudine and anti-microbial agents
for the treatment of microbial infections.
Inventors: |
Coates; Anthony (London,
GB), Hu; Yanmin (London, GB) |
Applicant: |
Name |
City |
State |
Country |
Type |
HELPERBY THERAPEUTICS LIMITED |
London |
N/A |
GB |
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Assignee: |
HELPERBY THERAPEUTICS LIMITED
(London, GB)
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Family
ID: |
50344128 |
Appl.
No.: |
15/111,275 |
Filed: |
January 29, 2015 |
PCT
Filed: |
January 29, 2015 |
PCT No.: |
PCT/GB2015/050209 |
371(c)(1),(2),(4) Date: |
July 13, 2016 |
PCT
Pub. No.: |
WO2015/114340 |
PCT
Pub. Date: |
August 06, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160331773 A1 |
Nov 17, 2016 |
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Foreign Application Priority Data
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Jan 30, 2014 [GB] |
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1401617.4 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/4525 (20130101); A61P 11/06 (20180101); A61P
31/08 (20180101); A61P 31/06 (20180101); A61P
13/08 (20180101); A61K 31/435 (20130101); A61P
25/00 (20180101); A61P 1/02 (20180101); A61P
35/00 (20180101); A61P 1/04 (20180101); A61K
31/496 (20130101); A61P 17/02 (20180101); A61K
31/7072 (20130101); A61K 38/12 (20130101); A61P
27/02 (20180101); A61P 31/04 (20180101); A61P
31/00 (20180101); A61P 17/00 (20180101); A61P
15/02 (20180101); A61P 31/10 (20180101); A61K
31/7072 (20130101); A61K 2300/00 (20130101); A61K
31/435 (20130101); A61K 2300/00 (20130101); A61K
31/496 (20130101); A61K 2300/00 (20130101); A61K
31/4525 (20130101); A61K 2300/00 (20130101); A61K
38/12 (20130101); A61K 2300/00 (20130101); Y02A
50/479 (20180101); Y02A 50/404 (20180101); Y02A
50/469 (20180101); Y02A 50/473 (20180101); Y02A
50/401 (20180101); Y02A 50/481 (20180101); Y02A
50/475 (20180101); Y02A 50/478 (20180101); Y02A
50/406 (20180101); Y02A 50/30 (20180101); Y02A
50/483 (20180101) |
Current International
Class: |
A61K
31/7072 (20060101); A61K 38/12 (20060101); A61K
31/435 (20060101); A61K 31/4525 (20060101); A61K
31/496 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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95100999 |
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Apr 1995 |
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WO |
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2000028074 |
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May 2000 |
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WO |
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2005014585 |
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Feb 2005 |
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WO |
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2006048747 |
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May 2006 |
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WO |
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2007054599 |
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Feb 2007 |
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WO |
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Not Cause a General Increase of Mutation Rates", Journal of
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an In Vitro pharmacokinetic/Pharmacodynamic Model", Antimicrobial
Agents and Chemotherapy, 57(8):3738-3745 (2013). cited by applicant
.
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0mg/ (Feb. 11, 2017). cited by applicant .
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aeruginosa Have Similar Resistance to Killing by Antimicrobials",
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resting bacterial populations", Proc. Natl. Acad. Sci. USA,
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Rifampin (RIF) Against COL-Resistant (R) and Susceptible (S)
KPC-producing Klebsiella pneumoniae (KPC-KP) clinical isolates",
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applicant .
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Staphylococcus aureus", Research Letters, 358(9277):207-208 (2001).
cited by applicant .
Van Asselt et al., "Detection of penicillin tolerance in
Streptococcus pyogenes", J. Med. Microbial., 38:197-202 (1993).
cited by applicant .
Pantopoulou et al., "Colistin offers prolonged survival in
experimental infection by multidrug-resistant Acinetobacter
baumannii: the significance of co-administration of rifampicin",
International Journal of Antimicrobial Agents, 29:51-55 (2007).
cited by applicant .
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combination with ciprofloxacin against Salmonella and Escherichia
coli", FEMS Immunology and Medical Microbiology, 7:23-28 (1993).
cited by applicant .
Keith et al., "In Vivo Efficacy of Zidovudine
(3'-Azido-S'-Deoxythymidine) in Experimental
Gram-Negative-Bacterial Infections", Antimicrobial Agents and
Chemotherapy, 33(4):479-483 (1989). cited by applicant .
Zhou et al., "Synergistic Antibiotic Combination Powders of
Colistin and Rifampicin Provide High Aerosolization Efficiency and
Moisture Protection", The AAPS Joumal, 16(1):37-47 (2014). cited by
applicant .
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nosocomial infections from multi-resistant Acinetobacter
baumannii", Journal of Infection, 53:274-278 (2006). cited by
applicant .
Bassetti et al, "Colistin and rifampicin in the treatment of
multidrug-resistant Acinetobacter baumannii infections", Journal of
Antimicrobial Chemotherapy, 61:417-420 (2008). cited by applicant
.
Bhardwaj et al., "Piperine, a Major Constituent of Black Pepper,
Inhibits Human P-glycoprotein and CYP3A4", The J. of Pharmacology
and Experimental Therapeutics, 302(2):645-650 (2002). cited by
applicant .
Chitnis et al., "In Vitro Synergistic Activity of Colistin with
Aminoglycosides, Beta-Lactams and Rifampin against
multidrug-resistant Gram-negative Bacteria", J. of Chemotherapy,
19(2):226-229 (2007). cited by applicant .
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ciprofloxacin, gentamicin and imipenem in vitro and in vivo", J. of
Antimicrobial Chemotherapy, 20:395-404 (1988). cited by applicant
.
Durante-Mangoni et al., "Colistin and Rifampicin Compared With
Colistin Alone for the Treatment of Serious Infections Due to
Extensively Drug-Resistant Acinetobacter baumannii: A Multicenter,
Randomized Clinical Trial", Colistin and Rifampicin Against
Acinetobacter, 57:349-358 (2013). cited by applicant .
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antimicrobial drugs", Nature Reviews, Drug Discovery, 1:895-910
(2002). cited by applicant .
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Microbiology, 13(1):34-40 (2005). cited by applicant .
Gonzales et al., "Infections due to vancomycin-resistant
Enterococcus faecium resistant to linezolid", Lancet,
357(9263):1179 (2001). cited by applicant.
|
Primary Examiner: Bradley; Christina
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
The invention claimed is:
1. A method of treating a bacterial infection, which comprises
administering to a mammal having said bacterial infection a
combination of: zidovudine; a polymyxin selected from colistin or
polymyxin B, or a pharmaceutically acceptable salt thereof; an
anti-tuberculosis antibiotic selected from rifampicin, rifapentine
and rifabutin; and optionally piperine, wherein the bacterial
infection is caused by Enterobacteriaceae.
2. A product comprising zidovudine in combination with: a polymyxin
selected from colistin and polymyxin B or a pharmaceutically
acceptable salt thereof; an anti-tuberculosis antibiotic selected
from rifampicin, rifapentine and rifabutin; and optionally
piperine; as a combined preparation for simultaneous, separate or
sequential use in killing clinically latent bacteria associated
with a bacterial infection, wherein the clinically latent bacteria
are Enterobacteriaceae.
3. A pharmaceutical composition comprising zidovudine in
combination with: a polymyxin selected from colistin and polymyxin
B or a pharmaceutically acceptable salt thereof; an
anti-tuberculosis antibiotic selected from rifampicin, rifapentine
and rifabutin; and optionally piperine; and a pharmaceutically
acceptable carrier for use in treating a bacterial infection,
wherein the bacterial infection is caused by
Enterobacteriaceae.
4. The method according to claim 1, wherein treating the bacterial
infection comprises killing clinically latent bacteria associated
with the bacterial infection, wherein the clinically latent
bacteria are Enterobacteriaceae.
5. The method according to claim 1, wherein zidovudine is
administered in combination with colistin and either rifampicin or
rifapentine.
6. A composition comprising zidovudine, colistin, and either
rifampicin or rifapentine for simultaneous, separate or sequential
use in treating a bacterial infection, wherein the bacterial
infection is caused by Enterobacteriaceae.
7. A method of treating a bacterial infection, which comprises
administering to a mammal having said bacterial infection a
combination of: zidovudine; colistin; and either rifampicin or
rifapentine, wherein the bacterial infection is caused by
Enterobacteriaceae.
8. The method according to claim 1 or 7, further comprising
administering piperine to said mammal.
9. The method according to claim 1 or claim 7, wherein the
bacterial infection is caused by E. coli, Proteus or
Klebsiella.
10. The method according to claim 1 or claim 7, wherein the
infection is caused by E. coli or Klebsiella.
11. The method according to claim 1 or claim 7, wherein the
infection is caused by a drug-resistant strain.
12. The method according to claim 11, wherein the infection is
caused by a carbapenemase-resistant strain or "extended spectrum
.beta.-lactamase" (ESPL) strain.
13. The method according to claim 1 or claim 7, for the treatment
of a urinary tract infection, nephritis, a kidney stone associated
infection or a catheter-associated infection caused by
Enterobacteriaceae.
14. The product according to claim 2, further comprising
piperine.
15. The pharmaceutical composition according to claim 3, further
comprising piperine.
16. The method according to claim 12, wherein the strain is New
Delhi Metallo-beta-lactamase-1 (NDM-1) resistant Klebs. Pneumonia.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application
No. PCT/GB2015/050209, filed on Jan. 29, 2015, which claims
priority from British Patent Application No. 1401617.4, filed on
Jan. 30, 2014, the contents of all of which are incorporated herein
by reference in their entirety.
TECHNICAL FIELD
The present invention relates to the use of an anti-retroviral
agent for killing clinically latent microorganisms associated with
microbial infections in combination with an anti-microbial
agent.
BACKGROUND
Before the introduction of antibiotics, patients suffering from
acute microbial infections (e.g. tuberculosis or pneumonia) had a
low chance of survival. For example, mortality from tuberculosis
was around 50%. Although the introduction of antimicrobial agents
in the 1940s and 1950s rapidly changed this picture, bacteria have
responded by progressively gaining resistance to commonly used
antibiotics. Now, every country in the world has
antibiotic-resistant bacteria. Indeed, more than 70% of bacteria
that give rise to hospital acquired infections in the USA resist at
least one of the main antimicrobial agents that are typically used
to fight infection (Nature Reviews, Drug Discovery, 1, 895-910
(2002)).
One way of tackling the growing problem of resistant bacteria is
the development of new classes of antimicrobial agents. However,
until the introduction of linezolid in 2000, there had been no new
class of antibiotic marketed for over 37 years. Moreover, even the
development of new classes of antibiotic provides only a temporary
solution, and indeed there are already reports of resistance of
certain bacteria to linezolid (Lancet, 357, 1179 (2001) and Lancet,
358, 207-208 (2001)).
In order to develop more long-term solutions to the problem of
bacterial resistance, it is clear that alternative approaches are
required. One such alternative approach is to minimise, as much as
is possible, the opportunities that bacteria are given for
developing resistance to important antibiotics. Thus, strategies
that can be adopted include limiting the use of antibiotics for the
treatment of non-acute infections, as well as controlling which
antibiotics are fed to animals in order to promote growth.
However, in order to tackle the problem more effectively, it is
necessary to gain an understanding of the actual mechanisms by
which bacteria generate resistance to antibiotic agents. To do this
requires first a consideration of how current antibiotic agents
work to kill bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a, FIG. 1b, and FIG. 1care time kill curves showing the
effect of the combination of colistin and rifampicin against NDM-1
Klebsiella pneumonia compared to colistin and rifampicin
singly.
FIG. 2a. FIG. 2b. FIG. 2c, and FIG. 2d are time kill curves showing
the effect of the combination of colistin and rifampicin against
NDM-1 E. coil compared to colistin and rifampicin singly.
FIG. 3 contains a plot of log CFU/ml for each treatment (i) to
(vi). Each treatment was tested, at 0 hour (left bar), 2 hours
(middle bar) and 6 hours (right bar) after administration of the
respective treatment.
FIG. 4 contains a plot of log CFU/ml for each treatment (i) to (vi)
at 0 hour, (left bar), 2 hours (middle bar) and 6 hours (right bar)
after administration of the respective treatment.
FIG. 5 is a table showing the results of a combination of
rifampicin and colistin with no addition of HT0120663. The wells
marked yellow demonstrate growth. Clear wells indicate growth
inhibition or no growth.
FIG. 6 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 0.5 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 7 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 1 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 8 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 2 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 9 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 4 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no Growth.
FIG. 10 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 8 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 11 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 16 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 12 is a table showing the results of a combination of
rifampicin and colistin with no addition of HT0120663. The wells
marked yellow demonstrate growth. Clear wells indicate growth
inhibition or no growth.
FIG. 13 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 0.125
.mu.g/ml. The wells marked yellow demonstrate growth. Clear wells
indicate growth inhibition or no growth.
FIG. 14 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 0.25
.mu.g/ml. The wells marked yellow demonstrate growth. Clear wells
indicate growth inhibition or no growth.
FIG. 15 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 0.5 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 16 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 1 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 17 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 2 .mu.g/ml,
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 18 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 4 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 19 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 3 .mu.g/ml.
Clear wells indicate growth inhibition or no growth.
FIG. 20 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 16 .mu.g/ml,
Clear wells indicate growth inhibition or no growth.
FIG. 21 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 32 .mu.g/ml.
Clear wells indicate growth inhibition or no growth.
FIG. 22 is a table showing the results of a combination of
rifampicin and colistin with no addition of HT0120663 against E.
coli NDM-1 2471. The wells marked yellow demonstrate growth. Clear
wells indicate growth inhibition or no growth.
FIG. 23 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 0.5 .mu.g/ml
against E. coli NDM-1 2471. The wells marked yellow demonstrate
growth. Clear wells indicate growth inhibition or no growth,
FIG. 24 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 1 .mu.g/ml
against E. coli NDM-1 2471. The wells marked yellow demonstrate
growth. Clear wells indicate growth inhibition or no growth.
FIG. 25 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 2 .mu.g/ml
against E. coli NDM-1 2471. The wells marked yellow demonstrate
growth. Clear wells indicate growth inhibition or no growth.
FIG. 26 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 4 .mu.g/ml
against E. coil NDM-1 2471. The wells marked yellow demonstrate
growth. Clear wells indicate growth inhibition or no growth.
FIG. 27 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 8 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 28 is a table showing the results of a combination of
rifampicin and colistin with addition of HT0120663 at 16 .mu.g/ml.
The wells marked yellow demonstrate growth. Clear wells indicate
growth inhibition or no growth.
FIG. 29 is a chequerboard showing synergy between colistin and
rifampicin against NDM-1 Klebsiella pneumonia.
FIG. 30 is a chequerboard showing synergy between colistin and
rifampicin against NDM-1 E. coli.
FIGS. 31 to 37 are each a chequerboard showing synergy between
colistin, rifampicin and HT0120663 against NDM-1 Klebsiella
pneumonia as described in Example 1.
FIGS. 38 to 43 are each a chequerboard showing synergy between
rifampicin and colistin against log phase gram negative bacteria as
described in Example 5.
DETAILED DESCRIPTION
Antimicrobial agents target essential components of bacterial
metabolism. For example, the .beta.-lactams (e.g. penicillins and
cephalosporins) inhibit cell wall synthesis, whereas other agents
inhibit a diverse range of targets, such as DNA gyrase (quinolones)
and protein synthesis (e.g. macrolides, aminoglycosides,
tetracyclines and oxazolidinones). The range of organisms against
which the antimicrobial agents are effective varies, depending upon
which organisms are heavily reliant upon the metabolic step(s) that
is/are inhibited. Further, the effect upon bacteria can vary from a
mere inhibition of growth (i.e. a bacteriostatic effect, as seen
with agents such as the tetracyclines) to full killing (i.e. a
bactericidal effect, as seen, e.g. with penicillin).
Bacteria have been growing on Earth for more than 3 billion years
and, in that time, have needed to respond to vast numbers of
environmental stresses. It is therefore perhaps not surprising that
bacteria have developed a seemingly inexhaustible variety of
mechanisms by which they can respond to the metabolic stresses
imposed upon them by antibiotic agents. Indeed, mechanisms by which
the bacteria can generate resistance include strategies as diverse
as inactivation of the drug, modification of the site of action,
modification of the permeability of the cell wall, overproduction
of the target enzyme and bypass of the inhibited steps.
Nevertheless, the rate of resistance emerges to a particular agent
has been observed to vary widely, depending upon factors such as
the agent's mechanism of action, whether the agent's mode of
killing is time- or concentration-dependent, the potency against
the population of bacteria and the magnitude and duration of the
available serum concentration.
It has been proposed (Science, 264, 388-393 (1994)) that agents
that target single enzymes (e.g. rifampicin) are the most prone to
the development of resistance. Further, the longer that suboptimal
levels of antimicrobial agent are in contact with the bacteria, the
more likely the emergence of resistance.
Moreover, it is now known that many microbial infections include
sub-populations of bacteria that are phenotypically resistant to
antimicrobials (J. Antimicrob. Chemother., 4, 395-404 (1988); J.
Med. Microbiol., 38, 197-202 (1993); J. Bacteriol., 182, 1794-1801
(2000); ibid. 182, 6358-6365 (2000); ibid. 183, 6746-6751 (2001);
FEMS Microbiol. Lett., 202, 59-65 (2001); and Trends in
Microbiology, 13, 34-40 (2005)). There appear to be several types
of such phenotypically resistant bacteria, including persisters,
stationary-phase bacteria, as well as those in the depths of
biofilms. However, each of these types is characterised by its low
rate of growth compared to log-phase bacteria under the same
conditions. Nutritional starvation and high cell densities are also
common characteristics of such bacteria.
Although resistant to antimicrobial agents in their slow-growing
state, phenotypically resistant bacteria differ from those that are
genotypically resistant in that they regain their susceptibility to
antimicrobials when they return to a fast-growing state (e.g. when
nutrients become more readily available to them).
The presence of phenotypically resistant bacteria in an infection
leads to the need for prolonged courses of antimicrobial agents,
comprising multiple doses. This is because the resistant, slowly
multiplying bacteria provide a pool of "latent" organisms that can
convert to a fast-growing state when the conditions allow (thereby
effectively re-initiating the infection). Multiple doses over time
deal with this issue by gradually killing off the "latent" bacteria
that convert to "active" form.
However, dealing with "latent" bacteria by administering prolonged
courses of antimicrobials poses its own problems. That is,
prolonged exposure of bacteria to suboptimal concentrations of
antimicrobial agent can lead to the emergence of genotypically
resistant bacteria, which can then multiply rapidly in the presence
of even high concentrations of the antimicrobial.
Long courses of antimicrobials are more likely to encourage the
emergence of genotypic resistance than shorter courses on the
grounds that non-multiplying bacterial will tend to survive and,
interestingly, probably have an enhanced ability to mutate to
resistance (Proc. Natl. Acad. Sci. USA, 92, 11736-11740 (1995); J.
Bacteriol., 179, 6688-6691 (1997); and Antimicrob. Agents
Chemother., 44, 1771-1777 (2000)).
In the light of the above, a new approach to combating the problem
of bacterial resistance might be to select and develop
antimicrobial agents on the basis of their ability to kill "latent"
microorganisms. The production of such agents would allow, amongst
other things, for the shortening of chemotherapy regimes in the
treatment of microbial infections, thus reducing the frequency with
which genotypical resistance arises in microorganisms.
The following articles disclose use of colistin, a polymixin, and
rifampicin as combination therapy for bacterial infections such as
multidrug resistant (MDR) Acinetobacter baumannii, (Motaouakkil S
et al J Infect (2006) 53 274-278, Bassetti M et al J Antimicrob
Chemo (2008) 61 417-420, Zhou A et al The AAPS J published online
on 16 Oct. 2013
DOI:10.1208/s12248-013-9537-8). Lee J J et al (Antimicrob Agents
& Chemo (2013) 57(8) 3738-3745) disclose the results of using a
combination of colistin and rifampicin in an in vitro model of
MDR-A. baumannii. Synergy was observed at come concentrations over
some time periods. A mechanism is proposed whereby colistin not
only self-promotes its own entry into the bacteria but thereby
increases the penetration by rifampicin.
Reporting on a multicentre, randomized clinical trial
Durante-Mangoni E et al Clin Infec Dis (2013) 57(3) 349-58 conclude
that due to there being no reduction in 30-day mortality in the
combination group, "These results indicate that, at present,
rifampicin should not be routinely combined with colistin in
clinical practice. The increased rate of A baumannii eradication
with combination treatment could still imply a clinical
benefit."
Tascini C et al (Antimicrob Agents & Chemo (2013) 57(8)
3990-3993 disclose the use of the same combination in
carbapenem-resistant Klebsiella pneumoniae. On the basis of the
favourable synergistic effect, the use of the combination in MDR-K
pneumonia is proposed as having a clinical role.
Recently, there has been report of an anti-retroviral drug,
zidovudine being active as an anti-microbial when combined with
gentamicin. Thus, Doleans-Jordheim A. et al., disclosed (Eur J Clin
Microbiol Infect Dis. 2011 October; 30(10):1249-56) that Zidovudine
(AZT) had a bactericidal effect on some enterobacteria, yet could
induce resistance in Escherichia coli. These resistances were
associated with various modifications in the thymidine kinase gene.
Furthermore, an additive or synergistic activity between AZT and
the two aminoglycoside antibiotics amikacin and gentamicin was
observed against enterobacteria.
Accordingly, in one embodiment of the present invention there is
provided the use of zidovudine in combination with; a polymyxin
selected from colistin or polymyxin B; an anti-tuberculosis
antibiotic selected from rifampicin, rifapentine or rifabutin; and
optionally piperine, for treating a microbial infection.
In a further embodiment the invention relates to a product
comprising zidovudine in combination with; a polymyxin selected
from colistin and polymyxin B; an anti-tuberculosis antibiotic
selected from rifampicin, rifapentine or rifabutin; and optionally
piperine, as a combined preparation for simultaneous, separate or
sequential use in killing clinically latent microorganisms
associated with a microbial infection.
An additional embodiment of the invention relates to a
pharmaceutical composition comprising zidovudine in combination
with; a polymyxin selected from colistin and polymyxin B; an
anti-tuberculosis antibiotic selected from rifampicin, rifapentine
or rifabutin; and optionally piperine, and a pharmaceutically
acceptable carrier for use in treating a microbial infection,
preferably killing clinically latent microorganisms associated with
a microbial infection.
In each described embodiment, the preferred anti-tuberculosis
antibiotic is rifampicin.
The present invention therefore further relates to a composition
comprising zidovudine, colistin and rifampicin for simultaneous,
separate or sequential use in treating a microbial infection.
The present invention therefore also includes;
The use of zidovudine for the treatment of a microbial infection in
combination with colistin and rifampicin or rifapentine,
The use of colistin for the treatment of a microbial infection in
combination with zidovudine and rifampicin or rifapentine, and,
The use of rifampicin or rifapentine for the treatment of a
microbial infection in combination with colistin and
zidovudine.
In each embodiment of the present invention the addition of
piperine is optional. On the basis of data regarding piperine as an
inhibitor of both human P-glycoprotein and CYP3A4 described in
inter alia, J Pharmacol Exp Ther (2002) 302(2) 645-650 the activity
of piperine is believed to be beneficial to the combinations
defined herein. Thus, in a preferred embodiment, piperine is
included in the combinations of the present invention.
The present invention is also based upon the unexpected finding
that the activity of the combinations of a polymyxin selected from
colistin and polymyxin B; and an anti-tuberculosis antibiotic
selected from rifampicin, rifapentine or rifabutin, is
substantially improved when administered with zidovudine. Moreover,
the combinations have surprisingly been shown to exhibit
synergistic antimicrobial activity against log phase (i.e.
multiplying) and/or clinically latent microorganisms. The
surprising biological activity of the combinations of the present
invention offers the opportunity to shorten chemotherapy regimes
and may result in a reduction in the emergence of microbial
resistance.
As described below, the combination of the present invention has
been demonstrated to be particularly effective against
drug-resistant bacteria opening the way for said combinations to be
administered both to drug-resistant strains and in said strains
before drug-resistance is built up i.e. as a first line
treatment.
The combinations of the present invention have in particular been
demonstrated to be effective against Gram-negative bacteria,
specifically drug-resistant Gram-negative bacteria.
As used herein, the term "in combination with" covers both separate
and sequential administration of an antimicrobial agent and an
anesthetic agent. When the agents are administered sequentially,
either agent may be administered first. When administration is
simultaneous, the agents may be administered either in the same or
a different pharmaceutical composition. Adjunctive therapy, i.e.
where one agent is used as a primary treatment and the other agent
is used to assist that primary treatment, is also an embodiment of
the present invention.
The combinations of the present invention may be used to treat
microbial infections. In particular they may be used to kill
multiplying and/or clinically latent microorganisms associated with
microbial infections. References herein to the treatment of a
microbial infection therefore include killing multiplying and/or
clinically latent microorganisms associated with such infections.
Preferably, the combinations of the present invention are used to
kill clinically latent microorganisms associated with microbial
infections.
As used herein, "kill" means a loss of viability as assessed by a
lack of metabolic activity.
As used herein, "clinically latent microorganism" means a
microorganism that is metabolically active but has a growth rate
that is below the threshold of infectious disease expression. The
threshold of infectious disease expression refers to the growth
rate threshold below which symptoms of infectious disease in a host
are absent.
The metabolic activity of clinically latent microorganisms can be
determined by several methods known to those skilled in the art;
for example, by measuring mRNA levels in the microorganisms or by
determining their rate of uridine uptake. In this respect,
clinically latent microorganisms, when compared to microorganisms
under logarithmic growth conditions (in vitro or in vivo), possess
reduced but still significant levels of: (I) mRNA (e.g. from 0.0001
to 50%, such as from 1 to 30, 5 to 25 or 10 to 20%, of the level of
mRNA); and/or (II) uridine (e.g. [.sup.3H]uridine) uptake (e.g.
from 0.0005 to 50%, such as from 1 to 40, 15 to 35 or 20 to 30% of
the level of [.sup.3H]uridine uptake).
Clinically latent microorganisms typically possess a number of
identifiable characteristics. For example, they may be viable but
non-culturable; i.e. they cannot typically be detected by standard
culture techniques, but are detectable and quantifiable by
techniques such as broth dilution counting, microscopy, or
molecular techniques such as polymerase chain reaction. In
addition, clinically latent microorganisms are phenotypically
tolerant, and as such are sensitive (in log phase) to the biostatic
effects of conventional antimicrobial agents (i.e. microorganisms
for which the minimum inhibitory concentration (MIC) of a
conventional antimicrobial is substantially unchanged); but possess
drastically decreased susceptibility to drug-induced killing (e.g.
microorganisms for which, with any given conventional antimicrobial
agent, the ratio of minimum microbiocidal concentration (e.g.
minimum bactericidal concentration, MBC) to MIC is 10 or more).
As used herein, the term "microorganisms" means fungi and bacteria.
References herein to "microbial", "antimicrobial" and
"antimicrobially" shall be interpreted accordingly. For example,
the term "microbial" means fungal or bacterial, and "microbial
infection" means any fungal or bacterial infection.
As summarised above, Doleans-Jordheim A. et al., disclosed (Eur J
Clin Microbiol Infect Dis. 2011 October; 30(10):1249-56) that
zidovudine (AZT) had a bactericidal effect on some enterobacteria,
in particular in combination with amikacin and gentamicin.
As used herein, the term "bacteria" (and derivatives thereof, such
as "microbial infection") includes, but is not limited to,
references to organisms (or infections due to organisms) of the
following classes and specific types:
Gram-positive cocci, such as Staphylococci (e.g. Staph. aureus,
Staph. epidermidis, Staph. saprophyticus, Staph. auricularis,
Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae,
Staph. cohnii cohnii, Staph. c. urealyticus, Staph. equorum, Staph.
gallinarum, Staph. haemolyticus, Staph. hominis hominis, Staph. h.
novobiosepticius, Staph. hyicus, Staph. intermedius, Staph.
lugdunensis, Staph. pasteuri, Staph. saccharolyticus, Staph.
schleiferi schleiferi, Staph. s. coagulans, Staph. sciuri, Staph.
simulans, Staph. warneri and Staph. xylosus);
Streptococci (e.g. beta-haemolytic, pyogenic streptococci (such as
Strept. agalactiae, Strept. canis, StrepL dysgalactiae
dysgalactiae, Strept. dysgalactiae equisimilis, Strept equi equi,
Strept equi zooepidemicus, Strept. iniae, Strept porcinus and
Strept pyogenes), microaerophilic, pyogenic streptococci
(Streptococcus "milleri", such as Strept. anginosus, Strept
constellatus constellatus, Strept constellatus pharyngidis and
Strept intermedius), oral streptococci of the "mitis"
(alpha-haemolytic--Streptococcus "viridans", such as Strept. mitis,
Strept. oralis, Strept. sanguinis, Strept. cristatus, Strept
gordonfi and Strept. parasanguinis), "salivarius" (non-haemolytic,
such as Strept. salivarius and Strept vestibularis) and "mutans"
(tooth-surface streptococci, such as Strept. criceti, Strept.
mutans, Strept ratti and Strept sobrinus) groups, Strept.
acidominimus, Strept. bovis, Strept. faecalis, Strept. equinus,
Strept. pneumoniae and Strept. suis, or Streptococci alternatively
classified as Group A, B, C, D, E, G, L, P, U or V
Streptococcus);
Gram-negative cocci, such as Neisseria gonorrhoeae, Neisseria
meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria
flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca,
Neisseria subflava and Neisseria weaveri;
Bacillaceae, such as Bacillus anthracis, Bacillus subtilis,
Bacillus thuringiensis, Bacillus stearothermophilus and Bacillus
cereus;
Enterobacteriaceae, such as Escherichia coli, Enterobacter (e.g.
Enterobacter aerogenes, Enterobacter agglomerans and Enterobacter
cloacae), Citrobacter (such as Citrob. freundii and Citrob.
divernis), Hafnia (e.g. Hafnia alvei), Erwinia (e.g. Erwinia
persicinus), Morganella morganii, Salmonella (Salmonella enterica
and Salmonella typhi), Shigella (e.g. Shigella dysenteriae,
Shigella flexneri, Shigella boydii and Shigella sonnei), Klebsiella
(e.g. Klebs. pneumoniae, Klebs. oxytoca, Klebs. ornitholytica,
Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs.
granulomatis (Calymmatobacterium granulomatis) and Klebs.
rhinoscleromatis), Proteus (e.g. Pr. mirabilis, Pr. rettgeri and
Pr. vulgaris), Providencia (e.g. Providencia alcalifaciens,
Providencia rettgeri and Providencia stuartii), Serratia (e.g.
Serratia marcescens and Serratia liquifaciens), and Yersinia (e.g.
Yersinia enterocolitica, Yersinia pestis and Yersinia
pseudotuberculosis);
Enterococci (e.g. Enterococcus avium, Enterococcus casseliflavus,
Enterococcus cecorum, Enterococcus dispar, Enterococcus durans,
Enterococcus faecalis, Enterococcus faecium, Enterococcus
flavescens, Enterococcus gallinarum, Enterococcus hirae,
Enterococcus malodoratus, Enterococcus mundtii, Enterococcus
pseudoavium, Enterococcus raffinosus and Enterococcus
solitarius);
Helicobacter (e.g. Helicobacter pylori, Helicobacter cinaedi and
Helicobacter fennelliae); Acinetobacter (e.g. A. baumanii, A.
calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. Iwoffi
and A. radioresistens);
Pseudomonas (e.g. Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas
maltophilia), Ps. alcaligenes, Ps. chlororaphis, Ps. fluorescens,
Ps. luteola. Ps. mendocina, Ps. monteilii, Ps. oryzihabitans, Ps.
pertocinogena, Ps. pseudalcaligenes, Ps. putida and Ps. stutzeri);
Bacteroides fragilis;
Peptococcus (e.g. Peptococcus niger);
Peptostreptococcus;
Clostridium (e.g. C. perfringens, C. difficile, C. botulinum, C.
tetani, C. absonum, C. argentinense, C. baratii, C. bifermentans,
C. beijerinckii, C. butyricum, C. cadaveris, C. camis, C. celatum,
C. clostridioforme, C. cochlearium, C. cocleatum, C. fallax, C.
ghonii, C. glycolicum, C. haemolyticum, C. hastiforme, C.
histolyticum, C. indolis, C. innocuum, C. irregulare, C. leptum, C.
limosum, C. malenominatum, C. novyi, C. oroticum, C.
paraputrificum, C. piliforme, C. putrefasciens, C. ramosum, C.
septicum, C. sordelii, C. sphenoides, C. sporogenes, C.
subterminale, C. symbiosum and C. tertium);
Mycoplasma (e.g. M. pneumoniae, M. hominis, M. genitalium and M.
urealyticum);
Mycobacteria (e.g. Mycobacterium tuberculosis, Mycobacterium avium,
Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium
kansasii, Mycobacterium chelonae, Mycobacterium abscessus,
Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium
africanum, Mycobacterium alvei, Mycobacterium asiaticum,
Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis,
Mycobacterium branderi, Mycobacterium brumae, Mycobacterium
celatum, Mycobacterium chubense, Mycobacterium confluentis,
Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium
flavescens, Mycobacterium gadium, Mycobacterium gastri,
Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium
goodii, Mycobacterium haemophilum, Mycobacterium hassicum,
Mycobacterium intracellulare, Mycobacterium interjectum,
Mycobacterium heidelberense, Mycobacterium lentiflavum,
Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium
microti, Mycobacterium mucogenicum, Mycobacterium neoaurum,
Mycobacterium nonchromogenicum, Mycobacterium peregrinum,
Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium
shimoidei, Mycobacterium simiae, Mycobacterium szulgai,
Mycobacterium terrae, Mycobacterium thermoresistabile,
Mycobacterium triplex, Mycobacterium triviale, Mycobacterium
tusciae, Mycobacterium ulcerans, Mycobacterium vaccae,
Mycobacterium wolinskyi and Mycobacterium xenopi);
Haemophilus (e.g. Haemophilus influenzae, Haemophilus ducreyi,
Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus
haemolyticus and Haemophilus parahaemolyticus);
Actinobacillus (e.g. Actinobacillus actinomycetemcomitans,
Actinobacillus equuli, Actinobacillus hominis, Actinobacillus
lignieresii, Actinobacillus suis and Actinobacillus ureae);
Actinomyces (e.g. Actinomyces israelii);
Brucella (e.g. Brucella abortus, Brucella canis, Brucella
melintensis and Brucella suis);
Campylobacter (e.g. Campylobacter jejuni, Campylobacter coli,
Campylobacter lari and Campylobacter fetus);
Listeria monocytogenes;
Vibrio (e.g. Vibrio cholerae and Vibrio parahaemolyticus, Vibrio
alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio
furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus
and Vibrio vulnificus);
Erysipelothrix rhusopathiae;
Corynebacteriaceae (e.g. Corynebacterium diphtheriae,
Corynebacterium jeikeum and Corynebacterium urealyticum);
Spirochaetaceae, such as Borrelia (e.g. Borrelia recurrentis,
Borrelia burgdorferi, Borrelia afzelii, Borrelia andersonii,
Borrelia bissettii, Borrelia garinii, Borrelia japonica, Borrelia
lusitaniae, Borrelia tanukii, Borrelia turdi, Borrelia valaisiana,
Borrelia caucasica, Borrelia crocidurae, Borrelia duttoni, Borrelia
graingeri, Borrelia hermsii, Borrelia hispanica, Borrelia
latyschewii, Borrelia mazzottii, Borrelia parkeri, Borrelia
persica, Borrelia turicatae and Borrelia venezuelensis) and
Treponema (Treponema pallidum ssp. pallidum, Treponema pallidum
ssp. endemicum, Treponema pallidum ssp. pertenue and Treponema
carateum);
Pasteurella (e.g. Pasteurella aerogenes, Pasteurella bettyae,
Pasteurella canis, Pasteurella dagmatis, Pasteurella gaffinarum,
Pasteurella haemolytica, Pasteurella multocida multocida,
Pasteurella multocida gallicida, Pasteurella multocida septica,
Pasteurella pneumotropica and Pasteurella stomatis);
Bordetella (e.g. Bordetella bronchiseptica, Bordetella hinzii,
Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis
and Bordetella trematum);
Nocardiaceae, such as Nocardia (e.g. Nocardia asteroides and
Nocardia brasiliensis); Rickettsia (e.g. Ricksettsii or Coxiella
burnetii);
Legionella (e.g. Legionalla anisa, Legionalla birminghamensis,
Legionalla bozemanii, Legionalla cincinnatiensis, Legionalla
dumoffii, Legionalla feeleii, Legionalla gormanii, Legionalla
hackeliae, Legionalla israelensis, Legionalla jordanis, Legionalla
lansingensis, Legionalla longbeachae, Legionalla maceachernii,
Legionalla micdadei, Legionalla oakridgensis, Legionalla
pneumophila, Legionalla sainthelensi, Legionalla tucsonensis and
Legionalla wadsworthii);
Moraxella catarrhalis;
Cyclospora cayetanensis;
Entamoeba histolytica;
Giardia lamblia;
Trichomonas vaginalis;
Toxoplasma gondii;
Stenotrophomonas maltophilia;
Burkholderia cepacia; Burkholderia mallei and Burkholderia
pseudomallei;
Francisella tularensis;
Gardnerella (e.g. Gardneralla vaginalis and Gardneralla
mobiluncus);
Streptobacillus moniliformis;
Flavobacteriaceae, such as Capnocytophaga (e.g. Capnocytophaga
canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis,
Capnocytophaga granulosa, Capnocytophaga haemolytica,
Capnocytophaga ochracea and Capnocytophaga sputigena);
Bartonella (Bartonella bacilliformis, Bartonella clarridgeiae,
Bartonella elizabethae, Bartonella henselae, Bartonella quintana
and Bartonella vinsonfi arupensis);
Leptospira (e.g. Leptospira biflexa, Leptospira borgpetersenii,
Leptospira inadai, Leptospira interrogans, Leptospira kirschneri,
Leptospira noguchii, Leptospira santarosai and Leptospira
weilii);
Spirillium (e.g. Spirillum minus);
Baceteroides (e.g. Bacteroides caccae, Bacteroides capillosus,
Bacteroides coagulans, Bacteroides distasonis, Bacteroides
eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides
merdae, Bacteroides ovatus, Bacteroides putredinis, Bacteroides
pyogenes, Bacteroides splanchinicus, Bacteroides stercoris,
Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides
uniformis, Bacteroides ureolyticus and Bacteroides vulgatus);
Prevotella (e.g. Prevotella bivia, Prevotella buccae, Prevotella
corporis, Prevotella dentalis (Mitsuokella dentalis), Prevotella
denticola, Prevotella disiens, Prevotella enoeca, Prevotella
heparinolytica, Prevotella intermedia, Prevotella loeschfi,
Prevotella melaninogenica, Prevotella nigrescens, Prevotella
oralis, Prevotella oris, Prevotella oulora, Prevotella tannerae,
Prevotella venoralis and Prevotella zoogleoformans);
Porphyromonas (e.g. Porphyromonas asaccharolytica, Porphyromonas
cangingivalis, Porphyromonas canoris, Porphyromonas cansulci,
Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas
crevioricanis, Porphyromonas endodontalis, Porphyromonas
gingivalis, Porphyromonas gingivicanis, Porphyromonas levii and
Porphyromonas macacae);
Fusobacterium (e.g. F. gonadiaformans, F. mortiferum, F. naviforme,
F. necrogenes, F. necrophorum necrophorum, F. necrophorum
fundiliforme, F. nucleatum nucleatum, F. nucleatum fusiforme, F.
nucleatum polymorphum, F. nucleatum vincentii, F. periodonticum, F.
russii, F. ulcerans and F. varium);
Chlamydia (e.g. Chlamydia trachomatis);
Cryptosporidium (e.g. C. parvum, C. hominis, C. canis, C. felis, C.
meleagridis and C. muris);
Chlamydophila (e.g. Chlamydophila abortus (Chlamydia psittaci),
Chlamydophila pneumoniae (Chlamydia pneumoniae) and Chlamydophila
psittaci (Chlamydia psittaci));
Leuconostoc (e.g. Leuconostoc citreum, Leuconostoc cremoris,
Leuconostoc dextranicum, Leuconostoc lactis, Leuconostoc
mesenteroides and Leuconostoc pseudomesenteroides);
Gemella (e.g. Gemella bergeri, Gemella haemolysans, Gemella
morbillorum and Gemella sanguinis); and
Ureaplasma (e.g. Ureaplasma parvum and Ureaplasma urealyticum).
Preferably, the bacterial infections treated by the combinations
described herein are gram-negative infections. Particular bacteria
that may be treated using a combination of the invention
include:
Gram positive bacteria;
Staphylococci, such as Staph. aureus (either Methicillin-sensitive
(i.e. MSSA) or Methicillin-resistant (i.e. MRSA)) and Staph.
epidermidis;
Streptococci, such as Strept. agalactiae and Strept. pyogenes;
Bacillaceae, such as Bacillus anthracis;
Enterococci, such as Enterococcus faecalis and Enterococcus
faecium; and
Gram negative bacteria;
Enterobacteriaceae, such as Escherichia coli, Klebsiella (e.g.
Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g. Pr.
mirabilis, Pr. rettgeri and Pr. vulgaris); Haemophilis
influenzae;
Mycobacteria, such as Mycobacterium tuberculosis.
Preferably, the bacterium is Enterobacteriaceae, such as
Escherichia coli, Klebsiella (e.g. Klebs. pneumoniae and Klebs.
oxytoca) and Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr.
vulgaris). The combination of the present invention is particularly
beneficial in treating (multi)-drug-resistant ((M)DR) bacteria.
With respect to Enterobacteriaceae, drug resistance most often
builds up to carbapenemase i.e. carbapenemase-resistant strains and
"extended spectrum .beta.-lactamase" (ESBL) strains for example New
Delhi Metallo-beta-lactamase-1 (NDM-1) resistant Klebs.
Pneumonia.
It should be kept in mind that although a combination such as that
claimed may initially be demonstrated to be functional in treating
(M)DR strains, they can then be used in treating non-resistant
strains. This is especially valuable in the context of the
presently claimed combination where the primary therapy for
Enterobacteriaceae, such as Escherichia coli, Klebsiella (e.g.
Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g. Pr.
mirabilis, Pr. rettgeri and Pr. vulgaris) are anti-microbial drugs
that are expensive due to prevailing patent protection. The
replacement of such "ethical" drugs by a combination of "generic"
antibiotics is thought to be beneficial from a therapeutic
perspective as well as financial/economic perspective in times
where governments are seeking to reduce the cost of healthcare.
The combinations of the present invention may be used to treat
infections associated with any of the above-mentioned bacterial
organisms, and in particular they may be used for killing
multiplying and/or clinically latent microorganisms associated with
such an infection.
Particular conditions which may be treated using the combination of
the present invention include tuberculosis (e.g. pulmonary
tuberculosis, non-pulmonary tuberculosis (such as tuberculosis
lymph glands, genito-urinary tuberculosis, tuberculosis of bone and
joints, tuberculosis meningitis) and miliary tuberculosis),
anthrax, abscesses, acne vulgaris, actinomycosis, asthma,
bacilliary dysentry, bacterial conjunctivitis, bacterial keratitis,
bacterial vaginosis, botulism, Buruli ulcer, bone and joint
infections, bronchitis (acute or chronic), brucellosis, burn
wounds, cat scratch fever, cellulitis, chancroid, cholangitis,
cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis,
nephritis, diffuse panbronchiolitis, diphtheria, dental caries,
diseases of the upper respiratory tract, eczema, empymea,
endocarditis, endometritis, enteric fever, enteritis, epididymitis,
epiglottitis, erysipelis, erysipclas, erysipeloid, erythrasma, eye
infections, furuncles, gardnerella vaginitis, gastrointestinal
infections (gastroenteritis), genital infections, gingivitis,
gonorrhoea, granuloma inguinale, Haverhill fever, infected burns,
infections following dental operations, infections in the oral
region, infections associated with prostheses, intraabdominal
abscesses, Legionnaire's disease, leprosy, leptospirosis,
listeriosis, liver abscesses, Lyme disease, lymphogranuloma
venerium, mastitis, mastoiditis, meningitis and infections of the
nervous system, mycetoma, nocardiosis (e.g. Madura foot),
non-specific urethritis, opthalmia (e.g. opthalmia neonatorum),
osteomyelitis, otitis (e.g. otitis externa and otitis media),
orchitis, pancreatitis, paronychia, pelveoperitonitis, peritonitis,
peritonitis with appendicitis, pharyngitis, phlegmons, pinta,
plague, pleural effusion, pneumonia, postoperative wound
infections, postoperative gas gangrene, prostatitis,
pseudo-membranous colitis, psittacosis, pulmonary emphysema,
pyelonephritis, pyoderma (e.g. impetigo), Q fever, rat-bite fever,
reticulosis, ricin poisoning, Ritter's disease, salmonellosis,
salpingitis, septic arthritis, septic infections, septicameia,
sinusitis, skin infections (e.g. skin granulomas, impetigo,
folliculitis and furunculosis), syphilis, systemic infections,
tonsillitis, toxic shock syndrome, trachoma, tularaemia, typhoid,
typhus (e.g. epidemic typhus, murine typhus, scrub typhus and
spotted fever), urethritis, urinary tract infections, wound
infections, yaws, aspergillosis, candidiasis (e.g. oropharyngeal
candidiasis, vaginal candidiasis or balanitis), cryptococcosis,
favus, histoplasmosis, intertrigo, mucormycosis, tinea (e.g. tinea
corporis, tinea capitis, tinea cruris, tinea pedis and tinea
unguium), onychomycosis, pityriasis versicolor, ringworm and
sporotrichosis; or infections with MSSA, MRSA, Staph. epidermidis,
Strept. agalactiae, Strept. pyogenes, Escherichia coli, Klebs.
pneumoniae, Klebs. oxytoca, Pr. mirabilis, Pr. rettgeri, Pr.
vulgaris, Haemophilis influenzae, Enterococcus faecalis and
Enterococcus faecium. In particular, the combination in kidney
stone associated infections and catheter-associated infections
arising from any of the bacteria described.
In a particular embodiment, the infection is selected from urinary
tract infections (cystitis, nephritis, kidney stone associated
infections and catheter-associated infections.
It will be appreciated that references herein to "treatment" extend
to prophylaxis as well as the treatment of established diseases or
symptoms.
Further preferred antimicrobial compounds for use in the present
invention are those capable of killing clinically latent
microorganisms. Methods for determining activity against clinically
latent bacteria include a determination, under conditions known to
those skilled in the art (such as those described in Nature
Reviews, Drug Discovery, 1, 895-910 (2002), the disclosures of
which are hereby incorporated by reference), of Minimum
Stationary-cidal Concentration ("MSC") or Minimum Dormicidal
Concentration ("MDC") for a test compound. A suitable compound
screening method against clinically latent microorganisms is
described in WO2000028074, the contents of which are incorporated
herein by reference as if the publication was specifically and
fully set forth herein.
As used herein the term "pharmaceutically acceptable derivative"
means: (a) pharmaceutically acceptable salts with either acids or
bases (e.g. acid addition salts); and/or (b) solvates (including
hydrates).
Acid addition salts that may be mentioned include carboxylate salts
(e.g. formate, acetate, trifluoroacetate, propionate, isobutyrate,
heptanoate, decanoate, caprate, caprylate, stearate, acrylate,
caproate, propiolate, ascorbate, citrate, glucuronate, glutamate,
glycolate, .alpha.-hydroxybutyrate, lactate, tartrate,
phenylacetate, mandelate, phenylpropionate, phenylbutyrate,
benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate,
methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate,
nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate,
suberate, sebacate, fumarate, malate, maleate, hydroxymaleate,
hippurate, phthalate or terephthalate salts), halide salts (e.g.
chloride, bromide or iodide salts), sulfonate salts (e.g.
benzenesulfonate, methyl-, bromo- or chloro-benzenesulfonate,
xylenesulfonate, methanesulfonate, ethanesulfonate,
propanesulfonate, hydroxyethanesulfonate, 1- or
2-naphthalene-sulfonate or 1,5-naphthalenedisulfonate salts) or
sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,
monohydrogenphosphate, dihydrogenphosphate, metaphosphate,
pyrophosphate or nitrate salts, and the like.
Compounds for use according to the invention may be administered as
the raw material but the active ingredients are preferably provided
in the form of pharmaceutical compositions.
The active ingredients may be used either as separate formulations
or as a single combined formulation. When combined in the same
formulation it will be appreciated that the two compounds must be
stable and compatible with each other and the other components of
the formulation.
Formulations of the invention include those suitable for oral,
parenteral (including subcutaneous e.g. by injection or by depot
tablet, intradermal, intrathecal, intramuscular e.g. by depot and
intravenous), rectal and topical (including dermal, buccal and
sublingual) or in a form suitable for administration by inhalation
or insufflation administration. The most suitable route of
administration may depend upon the condition and disorder of the
patient. In a preferred embodiment, the composition (administered
alone or separately) is administered systemically eg intravenously,
intramuscularly, via a catheter or inhaled.
Preferably, the compositions of the invention are formulated for
oral or topical administration. In a preferred embodiment, the
composition is a cream or an ointment adapted for nasal
administration, in particular for delivery to the anterior
nares.
The formulations may conveniently be presented in unit dosage form
and may be prepared by any of the methods well known in the art of
pharmacy e.g. as described in "Remington: The Science and Practice
of Pharmacy", Lippincott Williams and Wilkins, 21.sup.st Edition,
(2005). Suitable methods include the step of bringing into
association to active ingredients with a carrier which constitutes
one or more excipients. In general, formulations are prepared by
uniformly and intimately bringing into association the active
ingredients with liquid carriers or finely divided solid carriers
or both and then, if necessary, shaping the product into the
desired formulation. It will be appreciated that when the two
active ingredients are administered independently, each may be
administered by a different means.
When formulated with excipients, the active ingredients may be
present in a concentration from 0.1 to 99.5% (such as from 0.5 to
95%) by weight of the total mixture; conveniently from 30 to 95%
for tablets and capsules and 0.01 to 50% (such as from 3 to 50%)
for liquid preparations.
Formulations suitable for oral administration may be presented as
discrete units such as capsules, cachets or tablets (e.g. chewable
tablets in particular for paediatric administration), each
containing a predetermined amount of active ingredient; as powder
or granules; as a solution or suspension in an aqueous liquid or
non-aqueous liquid; or as an oil-in-water liquid emulsion or
water-in-oil liquid emulsion. The active ingredients may also be
presented a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with
one or more excipients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient in a
free-flowing form such as a powder or granules, optionally mixed
with other conventional excipients such as binding agents (e.g.
syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch,
polyvinylpyrrolidone and/or hydroxymethyl cellulose), fillers (e.g.
lactose, sugar, microcrystalline cellulose, maize-starch, calcium
phosphate and/or sorbitol), lubricants (e.g. magnesium stearate,
stearic acid, talc, polyethylene glycol and/or silica),
disintegrants (e.g. potato starch, croscarmellose sodium and/or
sodium starch glycolate) and wetting agents (e.g. sodium lauryl
sulphate). Moulded tablets may be made by moulding in a suitable
machine a mixture of the powdered active ingredient with an inert
liquid diluent. The tablets may be optionally coated or scored and
may be formulated so as to provide controlled release (e.g.
delayed, sustained, or pulsed release, or a combination of
immediate release and controlled release) of the active
ingredients.
Alternatively, the active ingredients may be incorporated into oral
liquid preparations such as aqueous or oily suspensions, solutions,
emulsions, syrups or elixirs. Formulations containing the active
ingredients may also be presented as a dry product for constitution
with water or another suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents (e.g. sorbitol syrup, methyl cellulose, glucose/sugar syrup,
gelatin, hydroxymethyl cellulose, carboxymethyl cellulose,
aluminium stearate gel and/or hydrogenated edible fats),
emulsifying agents (e.g. lecithin, sorbitan mono-oleate and/or
acacia), non-aqueous vehicles (e.g. edible oils, such as almond
oil, fractionated coconut oil, oily esters, propylene glycol and/or
ethyl alcohol), and preservatives (e.g. methyl or propyl
p-hydroxybenzoates and/or sorbic acid).
Topical compositions, which are useful for treating disorders of
the skin or of membranes accessible by digitation (such as membrane
of the mouth, vagina, cervix, anus and rectum), include creams,
ointments, lotions, sprays, gels and sterile aqueous solutions or
suspensions. As such, topical compositions include those in which
the active ingredients are dissolved or dispersed in a
dermatological vehicle known in the art (e.g. aqueous or
non-aqueous gels, ointments, water-in-oil or oil-in-water
emulsions). Constituents of such vehicles may comprise water,
aqueous buffer solutions, non-aqueous solvents (such as ethanol,
isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene
glycol, propylene glycol monolaurate, glycofurol or glycerol), oils
(e.g. a mineral oil such as a liquid paraffin, natural or synthetic
triglycerides such as Miglyol.TM., or silicone oils such as
dimethicone). Depending, inter alia, upon the nature of the
formulation as well as its intended use and site of application,
the dermatological vehicle employed may contain one or more
components selected from the following list: a solubilising agent
or solvent (e.g. a .beta.-cyclodextrin, such as hydroxypropyl
.beta.-cyclodextrin, or an alcohol or polyol such as ethanol,
propylene glycol or glycerol); a thickening agent (e.g.
hydroxymethyl cellulose, hydroxypropyl cellulose, carboxymethyl
cellulose or carbomer); a gelling agent (e.g. a
polyoxyethylene-polyoxypropylene copolymer); a preservative (e.g.
benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, a
benzoate, potassium sorbate or EDTA or salt thereof); and pH
buffering agent(s) (e.g. a mixture of dihydrogen phosphate and
hydrogen phosphate salts, or a mixture of citric acid and a
hydrogen phosphate salt). Topical formulations may also be
formulated as a transdermal patch.
Methods of producing topical pharmaceutical compositions such as
creams, ointments, lotions, sprays and sterile aqueous solutions or
suspensions are well known in the art. Suitable methods of
preparing topical pharmaceutical compositions are described, e.g.
in WO9510999, U.S. Pat. No. 6,974,585, WO2006048747, as well as in
documents cited in any of these references.
Topical pharmaceutical compositions according to the present
invention may be used to treat a variety of skin or membrane
disorders, such as infections of the skin or membranes (e.g.
infections of nasal membranes, axilla, groin, perineum, rectum,
dermatitic skin, skin ulcers, and sites of insertion of medical
equipment such as i.v. needles, catheters and tracheostomy or
feeding tubes) with any of the bacteria, fungi described above,
(e.g. any of the Staphylococci, Streptococci, Mycobacteria or
Pseudomonas organisms mentioned hereinbefore, such as S. aureus
(e.g. Methicillin resistant S. aureus (MRSA))).
Particular bacterial conditions that may be treated by topical
pharmaceutical compositions of the present invention also include
the skin- and membrane-related conditions disclosed hereinbefore,
as well as: acne vulgaris; rosacea (including
erythematotelangiectatic rosacea, papulopustular rosacea, phymatous
rosacea and ocular rosacea); erysipelas; erythrasma; ecthyma;
ecthyma gangrenosum; impetigo; paronychia; cellulitis; folliculitis
(including hot tub folliculitis); furunculosis; carbunculosis;
staphylococcal scalded skin syndrome; surgical scarlet fever;
streptococcal peri-anal disease; streptococcal toxic shock
syndrome; pitted keratolysis; trichomycosis axillaris; pyoderma;
external canal ear infections; green nail syndrome; spirochetes;
necrotizing fasciitis; Mycobacterial skin infections (such as lupus
vulgaris, scrofuloderma, warty tuberculosis, tuberculides, erythema
nodosum, erythema induratum, cutaneous manifestations of
tuberculoid leprosy or lepromatous leprosy, erythema nodosum
leprosum, cutaneous M. kansasii, M. malmoense, M. szulgai, M.
simiae, M. gordonae, M. haemophilum, M. avium, M. intracellulare,
M. chelonae (including M. abscessus) or M. fortuitum infections,
swimming pool (or fish tank) granuloma, lymphadenitis and Buruli
ulcer (Bairnsdale ulcer, Searles' ulcer, Kakerifu ulcer or Toro
ulcer)); as well as infected eczma, burns, abrasions and skin
wounds.
Compositions for use according to the invention may be presented in
a pack or dispenser device which may contain one or more unit
dosage forms containing the active ingredients. The pack may, e.g.
comprise metal or plastic foil, such as a blister pack. Where the
compositions are intended for administration as two separate
compositions these may be presented in the form of a twin pack.
Pharmaceutical compositions may also be prescribed to the patient
in "patient packs" containing the whole course of treatment in a
single package, usually a blister pack. Patient packs have an
advantage over traditional prescriptions, where a pharmacist
divides a patients' supply of a pharmaceutical from a bulk supply,
in that the patient always has access to the package insert
contained in the patient pack, normally missing in traditional
prescriptions. The inclusion of the package insert has been shown
to improve patient compliance with the physician's
instructions.
Suitable dosages and formulations for the administration of
colistin are described in the product label for Colomycin.RTM.
which can be found at
http://www.medicines.org.uk/emc/medicine/6301/SPC/Colomycin+Tablets/
Suitable dosages and formulations for the administration of
rifampicin are described in the product label for Rifadin.RTM.
capsules which can be found at
http://www.medicines.org.uk/emc/medicine/21223/SPC/Rifadin+300
mg+Capsules/ or Rifadin.RTM. for Infusion which can be found at
http://www.medicines.org.uk/emc/medicine/6435/SPC/Rifadin+For+Infusion+60-
0 mg/
Suitable dosages and formulations for the administration of
rifapentine are described in the product label for Priftin.RTM..
The preferred dosing regimen for the treatment of tuberculosis
caused by drug-susceptible organisms as part of regimens consisting
of an initial 2 month phase followed by a 4 month continuation
phase. The Initial Phase (2 Months) involves administration of 600
mg twice weekly for two months by direct observation of therapy,
with an interval of no less than 3 consecutive days (72 hours)
between doses, in combination with other antituberculosis drugs.
The Continuation Phase (4 Months) involves administration of 600 mg
once weekly for 4 months by direct observation therapy with
isoniazid or another appropriate antituberculous agent.
Suitable dosages and formulations for the administration of
zidovudine are described in the product label for Retrovir.RTM.
oral solution or capsules which can be found at
http://www.medicines.org.uk/emc/medicine/12444/SPC/Retrovir+250
mg+Capsules/
The administration of the combination of the invention by means of
a single patient pack, or patient packs of each composition,
including a package insert directing the patient to the correct use
of the invention is a desirable feature of this invention.
The individual components of the combination of the invention may
be administered simultaneously, separately or sequentially use. The
administering physician will be able to decide whether to utilise
the known dosing regimen and whether to maintain the simultaneous
administration. For example, the daily administration of zidovudine
and 8-hourly colistin may be superimposed on the regimen for
administering rifapentine or rifampicin, particularly
rifampicin.
According to a further embodiment of the present invention there is
provided a patient pack comprising at least one active ingredient
of the combination according to the invention and an information
insert containing directions on the use of the combination of the
invention.
In another embodiment of the invention, there is provided a double
pack comprising in association for separate administration, an
antimicrobial agent, preferably having biological activity against
clinically latent microorganisms, and an anesthetic agent,
preferably having biological activity against clinically latent
microorganisms.
The amount of active ingredients required for use in treatment will
vary with the nature of the condition being treated and the age and
condition of the patient, and will ultimately be at the discretion
of the attendant physician or veterinarian. In general however,
doses employed for adult human treatment will typically be in the
range of 0.02 to 5000 mg per day, preferably 1 to 1500 mg per day.
The desired dose may conveniently be presented in a single dose or
as divided doses administered at appropriate intervals, e.g. as
two, three, four or more sub-does per day.
Biological Tests
Test procedures that may be employed to determine the biological
(e.g. bactericidal or antimicrobial) activity of the active
ingredients include those known to persons skilled in the art for
determining: (a) bactericidal activity against clinically latent
bacteria; and (b) antimicrobial activity against log phase
bacteria.
In relation to (a) above, methods for determining activity against
clinically latent bacteria include a determination, under
conditions known to those skilled in the art (such as those
described in Nature Reviews, Drug Discovery 1, 895-910 (2002), the
disclosures of which are hereby incorporated by reference), of
Minimum Stationary-cidal Concentration ("MSC") or Minimum
Dormicidal Concentration ("MDC") for a test compound.
By way of example, WO2000028074 describes a suitable method of
screening compounds to determine their ability to kill clinically
latent microorganisms. A typical method may include the following
steps: (1) growing a bacterial culture to stationery phase; (2)
treating the stationery phase culture with one or more
antimicrobial agents at a concentration and or time sufficient to
kill growing bacteria, thereby selecting a phenotypically resistant
sub-population; (3) incubating a sample of the phenotypically
resistant subpopulation with one or more test compounds or agents;
and (4) assessing any antimicrobial effects against the
phenotypically resistant subpopulation.
According to this method, the phenotypically resistant
sub-population may be seen as representative of clinically latent
bacteria which remain metabolically active in vivo and which can
result in relapse or onset of disease.
In relation to (b) above, methods for determining activity against
log phase bacteria include a determination, under standard
conditions (i.e. conditions known to those skilled in the art, such
as those described in WO 2005014585, the disclosures of which
document are hereby incorporated by reference), of Minimum
Inhibitory Concentration ("MIC") or Minimum Bactericidal
Concentration ("MBC") for a test compound. Specific examples of
such methods are described below.
EXAMPLES
The chequerboard and Time kill experiments are described below and
in Antimicrob Chemo (2013) 68, 374-384.
Example 1; In Vitro Synergy Effect of Colistin, Rifampicin and
HT0120663 (Zidovudine) Against Log Phase NDM-1 Klebsiella pneumonia
BAA2471 by Three Dimensional Chequerboard Method
Objectives
To test the synergy effect of colistin, rifampicin and HT0120663
(zidovudine) against log phase NDM-1 Klebsiella pneumonia BAA2472
by chequerboard method
Materials and Methods
1. Bacterial strain used: NDM-1 BAA-2472TM, Klebsiella pneumoniae
was obtained from the American Type Culture Collection. 2. Growth
of bacteria: Log phase growth of BA2472 was carried out according
to SOP R-005-00 Log Phase Growth of Bacteria 3. Antibiotics and
preparation.
RMP was obtained from Sigma and was dissolved in DMSO to the stock
concentration of 10 mg/ml.
Colistin was obtained from Sigma (10 mg/ml).
HT0120663 was obtained from Sigma and was dissolved in DMSO to make
stock solution (10 mg/ml). 4. Chequerboard method
Rifampicin and colistin were combined using a two-dimensional
chequerboard with two-fold dilutions of each drug starting
concentration 8 .mu.g/ml for rifampicin and 4 .mu.g/ml for
colistin. The triple combinations (Colistin/rifampicin/HT0120663)
were tested by a three-dimensional chequerboard method where
HT0120663 was added at a single concentration on each plate at 0.5,
1, 2, 4, 8, 16 .mu.g/ml, respectively.
The overnight culture was diluted with nutrient broth (Oxoid) to
10.sup.5 CFU/ml and 280 .mu.l of the culture suspension was added
to each well to make the final volume of 300 .mu.l. 5. Incubation
of the compounds with the bacterial suspension was carried out for
24 hours. 6. The effects of combination were examined by
calculating the fractional inhibitory concentration index (FICI) of
double combination, as follows: (MIC of drug A, tested in
combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested
in combination)/(MIC of drug B, tested alone). The interaction of
the combination was defined as showing synergy if the FICI was
.ltoreq.0.5, no interaction if the FICI was >0.5 but <4.0 and
antagonism if the FICI was >4.0. Results
Combination of rifampicin and colistin with no addition of
HT0120663. The wells marked yellow in FIG. 5 demonstrate growth.
Clear wells indicate growth inhibition or no growth.
MIC of rifampicin was 4 .mu.g/ml. In combination with 0.5 .mu.g/ml
of colistin, MIC reduced to 0.25 .mu.g/ml. Colistin MIC was 1
.mu.g/ml. In combination with 2 .mu.g/ml of rifampicin, MIC reduced
to 0.125 .mu.g/ml. The FIC index is 0.188
Combination of rifampicin and colistin with addition of HT0120663
at 0.5 .mu.g/ml. The wells marked yellow in FIG. 6 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Addition of HT0120663 at 0.5 .mu.g/ml in rifampicin and colistin
combination reduced the MIC of rifampicin from 4 to 1 .mu.g/ml
which increased inhibition of growth.
Combination of rifampicin and colistin with addition of HT0120663
at 1 .mu.g/ml. The wells marked yellow in FIG. 7 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 2 .mu.g/ml. The wells marked yellow in FIG. 8 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 4 .mu.g/ml. The wells marked yellow in FIG. 9 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 1, 2 and 4 .mu.g/ml inhibited all the growth except
the last well where no drugs were present showing a synergistic
triple combination effect.
Combination of rifampicin and colistin with addition of HT0120663
at 8 .mu.g/ml. The wells marked yellow in FIG. 10 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 16 .mu.g/ml. The wells marked yellow in FIG. 11 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
When HT0120663 was increased to 8 .mu.g/ml or above, complete
inhibition of growth was seen. This was due to the MIC of HT0120663
(8 .mu.g/ml) which on its own inhibited the bacterial growth.
Summary and Conclusion
This data show that three drug combination increased potency of
each drug by reduction of MIC. Complete growth inhibition was seen
when HT0120663 was added at 8 .mu.g/ml or above.
Example 2: In Vitro Synergy Effect of Colistin, Rifampicin and
HT0120663 (Zidovudine) Against Log Phase NDM-1 Klebsiella pneumonia
BAA2473 by Chequerboard Method
Objectives
To test the synergy effect of colistin, rifampicin and HT0120663
(zidovudine) against log phase NDM-1 Klebsiella pneumonia BAA2473
by chequerboard method.
Materials and Methods
1. Bacterial strain used: NDM-1 BAA-2473TM, Klebsiella pneumoniae
was obtained from the American Type Culture Collection. 2. Growth
of bacteria: Log phase growth of BA2473 was carried out according
to SOP R-005-00 Log Phase Growth of Bacteria 3. Antibiotics and
preparation.
RMP was obtained from Sigma and was dissolved in DMSO to the stock
concentration of 10 mg/ml.
Colistin was obtained from Sigma (10 mg/ml).
HT0120663 was obtained from Sigma and was dissolved in DMSO to make
stock solution (10 mg/ml). 4. Chequerboard method--as described in
Example 1. (Colistin/rifampicin/HT0120663) were tested by a
three-dimensional chequerboard method where HT0120663 was added at
a single concentration on each plate.
The overnight culture was diluted with nutrient broth (Oxoid) to
10.sup.5 CFU/ml and 280 .mu.l of the culture suspension was added
to each well to make the final volume of 300 .mu.l. 5. Incubation
of the compounds with the bacterial suspension was carried out for
24 hours. 6. The effects of combination were examined by
calculating the fractional inhibitory concentration index (FICI) of
each combination, as follows: (MIC of drug A, tested in
combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested
in combination)/(MIC of drug B, tested alone). The interaction of
the combination was defined as showing synergy if the FICI was
.ltoreq.0.5, no interaction if the FICI was >0.5 but <4.0 and
antagonism if the FICI was >4.0. Results
Combination of rifampicin and colistin with no addition of
HT0120663. The wells marked yellow in FIG. 12 demonstrate growth.
Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 0.125 .mu.g/ml. The wells marked yellow in FIG. 13 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Addition of HT0120663 at 0.125 .mu.g/ml in rifampicin and colistin
combination showed no difference in inhibition of growth.
Combination of rifampicin and colistin with addition of HT0120663
at 0.25 .mu.g/ml. The wells marked yellow in FIG. 14 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Comparing with rifampicin and colistin combination only, addition
of HT0120663 at 0.25 .mu.g/ml reduced 4 fold of colistin MIC.
Combination of rifampicin and colistin with addition of HT0120663
at 0.5 .mu.g/ml. The wells marked yellow in FIG. 15 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 0.5 .mu.g/ml reduced 8 fold of colistin MIC.
Combination of rifampicin and colistin with addition of HT0120663
at 1 .mu.g/ml. The wells marked yellow in FIG. 16 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 2 .mu.g/ml. The wells marked yellow in FIG. 17 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 2 .mu.g/ml increased the effect of synergy by
reduction of rifampicin MIC.
Combination of rifampicin and colistin with addition of HT0120663
at 4 .mu.g/ml. The wells marked yellow in FIG. 18 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 4 .mu.g/ml increased the effect of synergy by
reduction of rifampicin MIC.
Combination of rifampicin and colistin with addition of HT0120663
at 8 .mu.g/ml. Clear wells in FIG. 19 indicate growth inhibition or
no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 16 .mu.g/ml. Clear wells in FIG. 20 indicate growth inhibition
or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 32 .mu.g/ml. Clear wells in FIG. 21 indicate growth inhibition
or no growth.
When HT0120663 was increased to 8 .mu.g/ml, complete inhibition of
growth was seen. This was due to the MIC of HT0120663 (8 .mu.g/ml)
which on its own inhibited the bacterial growth.
Summary and Conclusion
This data show that three drug combination increased potency of
each drug by reduction of MIC. Complete growth inhibition was seen
when HT0120663 was added at 8 .mu.g/ml or above.
Example 3; In Vitro Synergy Effect of Colistin, Rifampicin and
HT0120663 (Zidovudine) Against Log Phase NDM-1 Escherichia coli
BAA2471 by Three Dimensional Chequerboard Method
Objectives
To test the synergy effect of colistin, rifampicin and HT0120663
(zidovudine) against log phase NDM-1 E. coli BAA2471 by
chequerboard method.
Materials and Methods
1. Bacterial strain used: NDM-1 BAA-2471TM, E. coli was obtained
from the American Type Culture Collection. 2. Growth of bacteria:
Log phase growth of BA2471 was carried out according to SOP
R-005-00 Log Phase Growth of Bacteria 3. Antibiotics and
preparation.
RMP was obtained from Sigma and was dissolved in DMSO to the stock
concentration of 10 mg/ml.
Colistin was obtained from Sigma (10 mg/ml).
HT0120663 was obtained from Sigma and was dissolved in DMSO to make
stock solution (10 mg/ml). 4. Chequerboard method--as described in
Example 1.
The triple combinations (Colistin/rifampicin/HT0120663) were tested
by a three-dimensional chequerboard method where HT0120663 was
added at a single concentration on each plate at 0.5, 1, 2, 4, 8,
16 .mu.g/ml, respectively.
The overnight culture was diluted with nutrient broth (Oxoid) to
10.sup.5 CFU/ml and 280 .mu.l of the culture suspension was added
to each well to make the final volume of 300 .mu.l. 5. Incubation
of the compounds with the bacterial suspension was carried out for
24 hours. 6. The effects of combination were examined by
calculating the fractional inhibitory concentration index (FICI) of
double combination, as follows: (MIC of drug A, tested in
combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested
in combination)/(MIC of drug B, tested alone). The interaction of
the combination was defined as showing synergy if the FICI was
.ltoreq.0.5, no interaction if the FICI was >0.5 but <4.0 and
antagonism if the FICI was >4.0. Results
Combination of rifampicin and colistin with no addition of
HT0120663 against E. coli NDM-1 2471. The wells marked yellow in
FIG. 22 demonstrate growth. Clear wells indicate growth inhibition
or no growth.
MIC of rifampicin was 4 .mu.g/ml. In combination with 0.5 .mu.g/ml
of colistin, MIC reduced to 0.25 .mu.g/ml. Colistin MIC was 1
.mu.g/ml. In combination with 2 .mu.g/ml of rifampicin, MIC reduced
to 0.125 .mu.g/ml. The FIC index is 0.188
Combination of rifampicin and colistin with addition of HT0120663
at 0.5 .mu.g/ml against E. coli NDM-1 2471. The wells marked yellow
in FIG. 23 demonstrate growth. Clear wells indicate growth
inhibition or no growth.
Addition of HT0120663 at 0.5 .mu.g/ml in rifampicin and colistin
combination reduced the MIC of rifampicin from 4 to 1 .mu.g/ml
which increased inhibition of growth.
Combination of rifampicin and colistin with addition of HT0120663
at 1 .mu.g/ml against E. coli NDM-1 2471. The wells marked yellow
in FIG. 24 demonstrate growth. Clear wells indicate growth
inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 2 .mu.g/ml against E. coli NDM-1 2471. The wells marked yellow
in FIG. 25 demonstrate growth. Clear wells indicate growth
inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 1 and 2 .mu.g/ml inhibited all the growth except the
last two wells showing a synergistic triple combination effect.
Combination of rifampicin and colistin with addition of HT0120663
at 4 .mu.g/ml against E. coli NDM-1 2471. The wells marked yellow
in FIG. 26 demonstrate growth. Clear wells indicate growth
inhibition or no growth.
Comparing with rifampicin and colistin combination, addition of
HT0120663 at 4 .mu.g/ml inhibited all the growth except the last
well where no drugs were present showing a synergistic triple
combination effect.
Combination of rifampicin and colistin with addition of HT0120663
at 8 .mu.g/ml. The wells marked yellow in FIG. 27 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
Combination of rifampicin and colistin with addition of HT0120663
at 16 .mu.g/ml. The wells marked yellow in FIG. 28 demonstrate
growth. Clear wells indicate growth inhibition or no growth.
When HT0120663 was increased to 8 .mu.g/ml or above, complete
inhibition of growth was seen. This was due to the MIC of HT0120663
(8 .mu.g/ml) which on its own inhibited the bacterial growth.
Summary and Conclusion
This data show that three drug combination increased potency of
each drug by reduction of MIC. Complete growth inhibition was seen
when HT0120663 was added at 8 .mu.g/ml or above.
Example 4
Example 4.1: Colistin and Rifampicin Against NDM-1 Klebsiella
pneumonia
1.1 Chequerboard showing synergy between colistin and rifampicin
against NDM-1 Klebsiella pneumonia is provided in FIG. 29.
the effect of the combination of colistin and rifampicin against
NDM-1 Klebsiella pneumonia compared to colistin and rifampicin
singly.
Example 4.2: Colistin and Rifampicin Against NDM-1 Escherichia
coli
2.1 Chequerboard showing synergy between colistin and rifampicin
against NDM-1 E. coli is provided in FIG. 30.
2.2. FIG. 2 contains time kill curves FIG. 2(a), FIG. 2(b), FIG.
2(c) and FIG. 2(d) showing the effect of the combination of
colistin and rifampicin against NDM-1 E. coli compared to colistin
and rifampicin singly.
Example 4.3: Triple Combination (Rifampicin+Colistin+HT0120663
(Zidovudine))
HT0130001=Rifampicin
HT0130002=Colistin
HT0120663=Zidovudine
3. 1 Chequerboard showing synergy between colistin, rifampicin and
HT0120663 against NDM-1 Klebsiella pneumonia as described in
Example 1 is provided in FIGS. 31 to 37.
Example 5
Having demonstrated the effect of adding zidovudine to a
combination of colistin and rifampicin, the following examples
demonstrate synergy of the latter combination against a variety of
drug-resistant bacteria. On the basis of Examples 1-4, the addition
of zidovudine would again enhance the double combination of
colistin and rifampicin.
Experiment 5.1: In Vitro Synergy Effect of Colistin and Rifampicin
Against Log Phase Gram Negative Bacteria by Chequerboard Method
Objectives
To test the synergy effect of colistin and rifampicin against log
phase Gram negative bacteria including Escherichia coli and
Klebsiella-Enterobacter-Serratia group (KES group) by chequerboard
method
Materials and Methods
1. Bacterial strain used: Clinical antibiotic resistant Gram
negative isolates from St George's Hospital. NDM-1 strains were
obtained from the American Type Culture Collection BAA-2468TM,
Enterobacter cloacae. BAA-2469TM Escherichia coli. BAA-2470TM,
Klebsiella pneumoniae subsp. Pneumonia. BAA-2471TM, Escherichia
coli. BAA-2472TM, Klebsiella pneumoniae subsp. Pneumonia.
BAA-2473TM, Klebsiella pneumoniae and the National Collection of
Type Cultures from NCTC 13443, Klebsiella pneumonia 2. Growth of
bacteria: Log phase growth of BA2473 was carried out according to
SOP R-005-00 Log Phase Growth of Bacteria 3. Antibiotics and
preparation. i. RMP was obtained from Sigma and was dissolved in
DMSO to the stock concentration of 10 mg/ml. ii. Colistin was
obtained from Sigma (10 mg/ml).
Log phase bacterial culture was incubated with rifampicin and
colistin in combinations using chequerboard method.
The overnight culture was diluted with nutrient broth (Oxoid) to
10.sup.5 CFU/ml and 280 .mu.l of the culture was added to each well
to make the final volume of 300 .mu.l. 4. Incubation of the
compounds with the bacterial suspension was carried out for 24
hours. 5. The effects of combination were examined by calculating
the fractional inhibitory concentration index (FICI) of each
combination, as follows: (MIC of drug A, tested in
combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested
in combination)/(MIC of drug B, tested alone). The interaction of
the combination was defined as showing synergy if the FICI was
.ltoreq.0.5, no interaction if the FICI was >0.5 but <4.0 and
antagonism if the FICI was >4.0. Results
TABLE-US-00001 % of strains Number of FIC index (synergistic
Bacterial strains strains <0.5 >0.5 <2 >2 effect) E.
coli 56 55 1 0 98.2 Klebsiella-Enterobacter- 32 31 1 0 96.9
Serratia (KES group)
Summary and Conclusion
1. The synergistic combination of colistin and rifampicin showed in
98.2% of E. coli with FIC index less than 0.5.
2. The synergistic combination of colistin and rifampicin showed in
96.9% of the bacteria in KES group with FIC index less than
0.5.
3. Synergistic activities of colistin and rifampicin showed in all
NDM-1 strains.
Experiment 5.2: In Vitro Synergy Effect of Colistin and Rifampicin
Against Log Phase NDM-1 Strains by Chequerboard Method
Objectives
To test the synergy effect of colistin and rifampicin against log
phase NDM-1 strains by chequerboard method
Materials and Methods
1. Bacterial strain used: NDM-1 strains were obtained from the
American Type Culture Collection BAA-2468TM, Enterobacter cloacae.
BAA-2469TM Escherichia coli. BAA-2470TM, Klebsiella pneumoniae
subsp. Pneumonia. BAA-2471TM, Escherichia coll. BAA-2472TM,
Klebsiella pneumoniae subsp. Pneumonia. BAA-2473TM, Klebsiella
pneumoniae and the National Collection of Type Cultures from NCTC
13443, Klebsiella pneumonia 2. Growth of bacteria: Log phase growth
of BA2473 was carried out according to SOP R-005-00 Log Phase
Growth of Bacteria 3. Antibiotics and preparation. i. RMP was
obtained from Sigma and was dissolved in DMSO to the stock
concentration of 10 mg/ml. ii. Colistin was obtained from Sigma (10
mg/ml).
Log phase bacterial culture was incubated with rifampicin and
colistin in combinations using chequerboard method
The overnight culture was diluted with nutrient broth (Oxoid) to
10.sup.5 CFU/ml and 280 .mu.l of the culture was added to each well
to make the final volume of 300 .mu.l. 4. Incubation of the
compounds with the bacterial suspension was carried out for 24
hours. 5. The effects of combination were examined by calculating
the fractional inhibitory concentration index (FICI) of each
combination, as follows: (MIC of drug A, tested in
combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested
in combination)/(MIC of drug B, tested alone). The interaction of
the combination was defined as showing synergy if the FICI was
.ltoreq.0.5, no interaction if the FICI was >0.5 but <4.0 and
antagonism if the FICI was >4.0.
Results
The results are shown in FIGS. 38 to 43.
Summary and Conclusion
Colistin in combination with rifampicin showed FIC index less than
0.5 for BAA2468, BAA2469, BAA2470, BAA2471, BAA2472, BAA2473 and
NCTC13443 NDM-1 strains showing a significant synergistic
activity.
Example 6: In Vivo Synergy Effect of Colistin, Rifampicin and
HT0120663 (Zidovudine) Against NDM-1 E. coli in a Mouse Peritoneal
Infection Model 180314
Objectives
To investigate the activity of rifampicin, colistin and HT0120663
(zidovudine) in combination against NDM-1 E. coli in a mouse
peritoneal infection model.
Materials and Methods
1. Mice used: female Imprinting Control Region (ICR) mice aged 6 to
8 weeks were obtained from Harlan UK. 2. Bacterial culture used:
NDM-1 BAA2469 E. coli was obtained from the American Type Culture
Collection. 3. Drug preparation:
HT0120663 (zidovudine) solution was obtained from Pharmacy at the
concentration of 10 mg/ml.
Colistin used was Colomycin.RTM. (Forest Laboratories UK Ltd) which
was dissolved in water to 20 mg/ml.
Rifampicin used was Rifadin.RTM. (Sanofi-Aventis) 60 mg/ml. 4.
Mouse peritoneal infection model:
Overnight culture of NDM-1 BAA2469 E. coli (200 .mu.l) was injected
into the peritoneal cavities of the mice. 5. Drug
administration:
At 1.5 hours after infection, the triple combination
(colistin/rifampicin/HT0120663) was tested by intravenous
administration of rifampicin at 10 mg/kg, colistin at 20 mg/kg
and/or HT0120663 at 5 mg/kg singly or in combination to the
infected mice. The treatment combinations are shown in Table 1
below.
TABLE-US-00002 TABLE 1 mg/kg Colistin Rifampicin HT0120663 (i)
Colistin 20 0 0 (ii) Rifampicin 10 0 0 (iii) HT0120663 0 0 5 (iv)
Colistin + Rifampicin 20 10 0 (v) Colistin + Rifampicin + 20 10 5
HT0120663 (vi) Control 0 0 0
6. Organ CFU counting:
At 0 hour, 2 hours and 6 hours after administration of the above
treatments (i) to (vi), 1 ml of phosphate buffered saline (PBS) was
injected into the peritoneum of the mice followed by gently
massaging of the abdomen. Peritoneal fluid was then sampled
aseptically. The sampled fluid was diluted and CFU counts were
performed in order to determine the effect of the triple
combination (colistin/rifampicin/HT0120663).
The results are shown in FIG. 3.
Results
FIG. 3 contains a plot of log CFU/ml for each treatment (i) to
(vi). Each treatment was tested at 0 hour (left bar), 2 hours
(middle bar) and 6 hours (right bar) after administration of the
respective treatment.
Summary and Conclusion
1. Administration of rifampicin, colistin or HT0120663 alone showed
no in vivo activity against the NDM-1 E. coli. 2. At 2 hours after
treatment, there was no significant difference between rifampicin,
colistin or HT0120663 alone and the combinations. 3. At 6 hours
after treatment, the rifampicin and colistin combination (RMP/Col)
killed 4.5 log more bacteria than the single drugs. 4. At 6 hours
after treatment, the triple combination of
rifampicin/colistin/HT0120663 (RMP/Col/663) killed 4.5 log more
bacteria than rifampicin or colistin singly and 3 logs more than
HT0120663. The triple combination also killed more bacteria than
the double combination of RMP/Col.
Example 7: In Vivo Synergy Effect of Colistin, Rifampicin and
HT0120663 (Zidovudine) Against NDM-1 Klebsiella pneumonia in a
Mouse Peritoneal Infection Model 180314
Objectives
To investigate the activity of rifampicin, colistin and HT0120663
(zidovudine) in combination against NDM-1 Klebsiella pneumonia in a
mouse peritoneal infection model.
Materials and Methods
1. Mice used: female ICR mice aged 6 to 8 weeks were obtained from
Harlan UK. 2. Bacterial cultures used: NDM-1 BAA2470 K. pneumoniae
obtained from the American Culture Collection. 3. Drug
preparation:
HT0120663 solution was obtained from Pharmacy at the concentration
of 10 mg/ml. Colistin used was Colomycin.RTM. (Forest Laboratories
UK Ltd) which was dissolved in water to 20 mg/ml.
Rifampicin used was Rifadin.RTM. (Sanofi-Aventis) 60 mg/ml. 4.
Mouse peritoneal infection model:
Overnight culture of NDM-1 BAA2470 K. pneumoniae (200 .mu.l) was
injected into the peritoneal cavities of the mice. 5. Drug
administration
At 1.5 hours after infection, the triple combination
(colistin/rifampicin/HT0120663) was tested by intravenous
administration of rifampicin at 10 mg/kg, colistin at 20 mg/kg
and/or HT0120663 at 5 mg/kg singly or in combination to the
infected mice as shown in Table 2.
TABLE-US-00003 TABLE 2 mg/kg Colistin Rifampicin HT0120663 (i)
Colistin 20 0 0 (ii) Rifampicin 10 0 0 (iii) HT0120663 0 0 5 (iv)
Colistin + Rifampicin 20 10 0 (v) Colistin + Rifampicin + 20 10 5
HT0120663 (vi) Control 0 0 0
6. Organ CFU counting:
At 0 hour, 2 and 6 hours after administration of the above
treatments (i) to (vi), 1 ml of phosphate buffered saline (PBS) was
injected into the peritoneum of the mice followed by gently
massaging of the abdomen. Peritoneal fluid was then sampled
aseptically. The sampled fluid was diluted and CFU counts were
performed in order to determine the effect of the triple
combination (colistin/rifampicin/HT0120663).
The results are shown in FIG. 4.
Results
FIG. 4 contains a plot of log CFU/ml for each treatment (i) to (vi)
at 0 hour, (left bar), 2 hours (middle bar) and 6 hours (right bar)
after administration of the respective treatment.
Summary and Conclusions
1. Administration of rifampicin, colistin or HT0120663 alone showed
no in vivo activity against the NDM-1 K. pneumoniae. 2. At 2 hours
after treatment, there was no significant difference between the
colistin and rifampicin combination or colistin and HT0120663,
singly or in combination. However, the triple combination
(rifampicin/colistin/HT0120663) reduced 1 log of initial inoculum.
3. At 6 hours after treatment, the rifampicin and colistin
combination killed 3.1 log more bacteria than the single drugs. 4.
At 6 hours after treatment, the colistin and HT0120663 combination
killed 4.1 log more bacteria than each single drug. 5. At 6 hours
after treatment, the triple combination of
rifampicin/colistin/HT0120663 killed 4.6 log more bacteria than
rifampicin, colistin or HT0120663 singly and also more bacteria
than the double combinations of rifampicin/colistin and
colistin/HT0120663.
Faced with the challenge of improving anti-microbial therapy in
view of the increase in multidrug resistant strains, the Examples
demonstrate a significant effect of adding zidovudine to a
therapeutic regimen consisting of colistin and an anti-tuberculosis
antibiotic such as rifampicin or rifapentine or rifabutin. For the
first time, the addition of zidovudine has been shown to have a
synergisitc effect on this regimen. This triple combination may
therefore offer a significant improvement to the treatment of
anti-microbial infections arising from a range of bacteria in
addition to those utilised in the Examples.
* * * * *
References